Targeting the Sonic Hedgehog-Gli1 Pathway as a Potential New Therapeutic Strategy for Myelodysplastic Syndromes

<div><p>The complex mechanistic array underlying the pathogenesis of myelodysplastic syndrome (MDS) is still unclear. Although dysregulations of different signaling pathways involved in MDS have been described, the identification of specific biomarkers and therapy targets remains an important task in order to establish novel therapeutic approaches. Here, we demonstrated that the Shh signaling pathway is active in MDS and correlated it with disease progression. Additionally, the knockdown of Gli1 significantly inhibited cell proliferation <i>in vitro</i> and <i>in vivo</i>. Gli1 silencing also induced apoptosis and G0/G1 phase arrest. Furthermore, Gli1 silencing enhanced the demethylating effect of 5-aza-2'-deoxycytidine on the p15 gene promoter and subsequently promoted its expression by inhibiting DNA methyltransferase 1(DNMT1). Our findings show that the Shh signaling pathway plays a role in the pathogenesis and disease progression of MDS, and proceeds by modulating DNA methylation. This pathway may prove to be a potential therapeutic target for enhancing the therapeutic effects of 5-azacytidine on malignant transformation of MDS.</p></div

Gli1 silencing enhanced the demethylating effect of 5-aza-dC on p15 and subsequently promoted p15 expression.

<p>(A). Relative expression of DNMT1 and p15 mRNA upon Gli1 knockdown was analyzed by real-time PCR. Expression levels of beta-actin (ACTB), used as a housekeeping gene, were taken as control. Fold-change was calculated with the 2^−ΔΔCt method compared with controls. All experiments were performed in triplicate and the results are expressed as average ± SEM. *P < 0.05. (B). Western blot analysis of the effects of Gli1 knockdown on DNMT1 and p15 protein expression. Each sample was normalized to the respective related beta-actin (ACTB) expression. (C). Methylation-specific PCR for identification of changes in the methylation status of p15 promoter in MUTZ-1 cells transfected with sh-Gli1 (Gli1 sh1 and Gli1 sh2) and control lentiviral vector (Scramble) treated with or without 5-aza-dC (2 μM) for 48h. Images represent PCR-amplified products separated on 2% agarose gels and visualized under UV light after staining with ethidium bromide.</p

The characteristics of patients.

<p>RA, refractory anemia; RARS, refractory anemia with ringed sideroblasts; RCMD, refractory cytopenia with multilineage dysplasia; RAEB, RA with excess blasts. MDS, myelodysplastic syndrome; AML, acute myelocytic leukemia; WHO, world health organization; IPSS, International Prognostic Scoring System.</p><p>The characteristics of patients.</p

Gli1 predicts poor prognosis in AML and Shh-Gli1 signaling is activated in MDS.

<p>(A). Kaplan-Meier plots of overall survival (OS) in patients with AML, stratified by Gli1 expression. Data was obtained from the Cancer Genome Atlas (TCGA) database [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0136843#pone.0136843.ref023" target="_blank">23</a>] (r = 1.41, P = 0.075). (B). Real-time PCR analysis of DNMT1, Gli1, Smo, and Shh in primary CD34⁺cells (n = 32) from MDS and acute myeloid leukemia associated with MDS marrow samples. (C). A statistically significant positive correlation of DNMT1 and Gli1 expression in MDS (Spearman’s correlation analysis, R = 0.563, P < 0.01). (D). Immunofluorescent staining of SHH and GLI1 in primary bone marrow-derived CD34⁺cells from MDS patients and control healthy donors.</p

Primer sequences for real-time PCR.

<p>Primer sequences for real-time PCR.</p

Knockdown of Gli1 inhibits <i>in vitro</i> and <i>in vivo</i> MDS cell growth.

<p>(A). CCK8 assays were performed to determine the proliferation of MUTZ-1 cells transfected with sh-Gli1 (Gli1 sh1 and Gli1 sh2) and control lentiviral vector (Scramble), wherein the knockdown of Gli1 was found to suppress MUTZ-1 growth <i>in vitro</i>. Two-way ANOVA with post hoc analysis (Bonferroni test) was performed to determine the effects of Gli1 knockdown and multiple time points on cell viability. (B). Representative images of tumor growth in nude mice. (C). Effects of Gli1 knockdown on tumor growth <i>in vivo</i>, wherein tumors developed from MUTZ-1 cells stably transfected with shGli1 showed smaller tumor volume and weights. *P < 0.05. Results are expressed as mean ± SEM.</p

Patients characteristics.

<p>Patients characteristics.</p

Relapsed patients characteristics.

<p>M: male; F: female; H: hematologic relapse; E: isolated extramedullary relapse; Y: yes; N: no.</p><p>Relapsed patients characteristics.</p

Calculation of peak area in chimerism.

<p>The first line is the samples of the donor. The second line is the samples of the recipient prior to transplantation. The third line is the samples examined following transplantation. The fourth line is the calculation formula of peak area in chimerism.</p

Characteristics of the B-ALL patients who experienced hematologic relapse.

<p>data weren’t monitored BM: unfractionated cells of bone marrow.</p><p>Characteristics of the B-ALL patients who experienced hematologic relapse.</p